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Pack - 15 Flashcards

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AP® Biology flashcards Biology 101 flashcards
1
Q

What are the risks of injecting a human donor with animal insulin? (2)

A

Rejection by the immune system.

Risk of infection.

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2
Q

What is recombinant DNA?

A

The combining of DNA from two different organisms. (That can be from different species).

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3
Q

What is a transgenic organism? What is is also known as?

A
  • Has had its genome modified (by the process of recombinant DNA)
  • Genetically modified organisms.
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4
Q

Why is it that DNA is not only accepted by another species but also functions normally when transferred?

A

• Genetic code is universal.

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5
Q

Why can proteins still be made from DNA recombinant DNA in another organism?

A

• The processes of transcription and translation are effectively the same in all organisms.

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6
Q

Describe the 5 stages of making a protein using DNA technology of gene transfer and cloning. Describe each stage.

A
  • Isolation - of DNA fragments that have the gene fir the desired protein.
  • Insertion - of DNA fragment into a vector.
  • Transformation - transfer of DNA into a suitable host cell.
  • Identification - of host cells that have successfully taken up the gene by gene markers.
  • growth/cloning of the population.
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7
Q

Give three ways of producing specific gene fragments.

A
  • Conversion of mRNA to cDNA using reverse transcriptase.
  • Using restriction endonucleases to cut fragments containing the desired gene.
  • A gene machine - based on a known protein structure.
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8
Q

Why does reverse transcriptase have its name? Where is it found?

A
  • It catalyses the production of DNA from RNA (opposite of transcription).
  • Retroviruses e.g. HIV
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9
Q

Describe how reverse transcriptase can be used to isolate a gene. (4 steps)

A
  • mRNA is extracted from a cell that produces lots of this mRNA for the desired gene.
  • mRNA acts as a template which complementary (cDNA) is formed using reverse transcriptase.
  • Hydrolysis of the two strands using an enzyme producing single stranded cDNA
  • DNA polymerase is used to from double stranded DNA using the cDNA as template.
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10
Q

Where are restriction endonucleases found and what is their role originally?

A

Bacteria- to cut up viral DNA

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11
Q

What does a restriction endonuclease do?

A

Cuts double stranded DNA at a specific base sequence called a recognition sequence.

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12
Q

What is a recognition sequence?

A

Where restriction endonucleases cut DNA.

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13
Q

What are blunt ends?

A

When restriction endonucleases cut DNA between two opposite base pairs.

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14
Q

What are sticky ends?

A

When a restriction endonuclease cuts DNA at different points on each strand leaving exposed single stranded DNA.

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15
Q

What is a palindromic recognition sequence?

A

The recognition sequence reads the same in both directions.

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16
Q

In 8 steps how can a gene machine produce a gene fragment for a desired gene.

A
  • Base sequence determined. (from amino acid)
  • Sequence is checked.
  • The computer designs a series of small overlapping single strands of nucleotides - oligonucleotides.
  • Each of the oligonucleotides is assembled in an automated process - one nucleotide at a time.
  • Oligonucleotides are joined together (no introns)
  • Polymerase chain reaction to multiply the gene and construct complementary strand.
  • Inserted into a plasmid using sticky ends.
  • Genes are checked.
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17
Q

Why is the gene sequence checked before being manufactured by a gene machine?

A

Biosecurity, biosafety and ethical requirements.

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18
Q

What is the advantage of using a gene machine to produce DNA fragments?

A
  • Any sequence of nucleotides can be produced in a very short amount of time.
  • Accurate
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19
Q

Why is it useful for a gene to be free of introns when produced?

A

• Can be transferred into prokaryotic cells and still be translated.

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20
Q

In what two ways can a DNA fragment be cloned?

A
  • In vivo - transferring the fragments into a host cell using a vector.
  • In vitro - PCR
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21
Q

What is the name of the DNA sequence that restriction endonucleases attach to?

A

Recognition site.

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22
Q

What is the importance of using sticky ends?

A

Provided the same restriction endonuclease is used, you can combine DNA of one organism with that of any other.

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23
Q

What is the advantage of using sticky ends over blunt ends?

A

The restriction endonuclease leaves a short sequence of single stranded DNA. This is complementary if the same enzyme is used.

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24
Q

What does DNA ligase do?

A

Joins the phosphodiester bonds between the two joined sections of DNA.

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25
Q

How two sticky ends from the same enzyme attach?

A

Complementary base pairing. Then DNA ligase.

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26
Q

What needs to be added to a DNA fragment to be cloned (in vivo) before insertion into a vector?

A

A promoter - before

A terminator region - after.

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27
Q

What is the purpose of the promoter region of a gene? (2) What does it allow to occur?

A
  • Where RNA polymerase must attach.
  • Where transcription factors attach.
  • Transcription begins
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28
Q

How does RNA polymerase bind to DNA of a gene?

A

Nucleotide sequence of the promoter region.

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29
Q

What is the purpose of the terminator region of a gene? What does it terminate?

A
  • RNA polymerase detaches

* Transcription

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30
Q

What is the purpose of inserting target DNA into a vector?

A

Vector is used to transport the DNA into the host cell.

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31
Q

What is the most common vector used in in vivo cloning?

A

Plasmids

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32
Q

What is plasmid?

A

Circular length DNA found in prokaryotic cells. Separate from the main bit of DNA.

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33
Q

What type of gene do plasmids almost always contain?

A

Antibiotic resistance gene.

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34
Q

Where do restriction endonucleases cut the vector?

A

In the antibiotic resistance gene.

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35
Q

Why do you need to cut the vector with the same restriction endonuclease as the one used to cut the DNA fragment?

A

So the sticky ends of the opened up plasmid form complementary base pairs with the sticky ends of the DNA fragment.

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36
Q

How are plasmids joined to DNA fragments permanently?

A

DNA ligase forms phsophodiester bonds.

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37
Q

What is transformation?

A

Transferal of recombinant plasmids into the bacterial cells.

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38
Q

Outline the 5 steps of gene transfer and in vivo cloning.

A
  • Isolation of DNA with required gene.
  • Insertion into vector using DNA ligase.
  • Transformation into host cell.
  • Identification of cells that have taken up the recombinant DNA - markers.
  • Growth/cloning.
39
Q

What conditions are used for transformation of the vector into the host cell? (2) How does this work? (1)

A
  • Heat shock

* Calcium ions - make the cell surface membrane permeable so vectors enter.

40
Q

Why might not all bacterial cells posses the required gene’s DNA after transformation? (3)

A
  • Only about 1% of bacterial cells take up the plasmids when mixed together.
  • Some plasmids will have self ligated without DNA fragment.
  • Some DNA fragments form loops (“plasmids”) by themselves.
41
Q

Describe in 4 steps the process of testing whether bacteria have taken up a specific plasmid (with recombinant DNA or NOT) containing the ampicillin resistance gene.

A
  • Bacteria are grown on a medium containing ampicillin.
  • Bacterial cells that have taken up the plasmid will be resistant.
  • These will survive as they can break down ampicillin.
  • The bacterial cells without the plasmids will have no gene and will die.
42
Q

What is the issue with using only one antibiotic resistance gene to show whether bacteria have taken up a plasmid?

A

Some bacteria will take up the plasmid without the new gene (self ligated) and so they will survive.

43
Q

What can be done to identify which bacteria contain the target gene once you have identified which bacteria contain the plasmid (with or without the target gene)?

A

• Use a marker gene e.g. a second antibiotic resistance gene.

44
Q

Name three possible types of marker gene.

A
  • Antibiotic resistance (a second one)
  • Fluorescent proteine gene
  • Enzyme whose action can be identified.
45
Q

Describe how two antibiotic resistance genes can be used to identify which bacteria have taken up the target gene. (6)

A
  • Cut open the plasmid (restriction endonuclease and sticky ends - same as the one that cut DNA) in the middle of antibiotic resistance A gene.
  • DNA forms H-bonds with this plasmid. DNA ligase.
  • Gene A no longer works.
  • All plasmids contain gene B so grown on a culture of gene B to get rid of those that have taken up no plasmid.
  • Replica plate.
  • Grow on a culture that contains gene A to see which bacteria die (these are the target bacteria.)
46
Q

What is replica plating.

A

Copying colonies of bacteria onto a second plate so when bacteria on one plate die due to an antibiotic these can be identified on the other plate.

47
Q

A target gene is inserted in the middle of a fluorescent gene in a plasmid. How can this marker be used to identify bacteria with the target gene. Why is this better than two antibiotic resistance genes?

A
  • Those that do not fluoresce have the target gene.

* No need for replica plating as target cells don’t die. Therefore faster.

48
Q

How is an enzyme gene marker used to identify bacteria that have taken up the required gene?

A
  • Insert target gene into the middle of enzyme gene.
  • Enzyme gene doesn’t function.
  • Add substrate.
  • Colonias that don’t change colour contain the target gene.
49
Q

What components does the polymerase chain reaction require? (5)

A
  • the DNA fragment to be copied
  • taq DNA polymerase
  • Primers
  • Nucleotides.
  • Thermocylcer - computer controlled machine.
50
Q

What is taq DNA polymerase? Why is it used in the PCR?

A
  • From bacteria in hot springs.

* Doesn’t denature a the high temperatures during PCR.

51
Q

What is a primer? Why is it necessary in PCR?

A
  • Short sequence of nucleotides that has set bases complementary to those at the end of the DNA fragment.
  • DNA polymerase needs a primer to bind to to begin replication. It also prevents the two strands rejoining.
52
Q

What are the three steps of the PCR? What happens in each? What temp. (approx.)? (3)

A
  • Separation of DNA strands - 95C - Two strands separate - H-bonds break.
  • Addition of primers - 55C - primers anneal to the end of base sequence - starting sequence for DNA polymerase - prevent two strands rejoining.
  • Synthesis of DNA - 72C - optimum temperature for taq DNA polymerase - add complementary nucleotides to both strands to form two new DNA molecule.
  • Repeat
53
Q

At what rate does the number of DNA strands grow at in PCR?

A

Exponentially 2^n. N is the tuber of complete cycles.

54
Q

Give two advantages of in vitro cloning.

A
  • Extremely rapid. (100 billion DAN fragments in hours)

* Does no require living cells - no culturing.

55
Q

What can PCR be used for that in vivo cloning is not used for? Why?

A

• Forensics - amplifying DNA at crime scene. Very quick

56
Q

What is a potential problem with amplifying a tiny amount of DNA found at a crime scene using PCR?

A

• Contaminating DNA will be greatly amplified as well.

57
Q

Give 5 advantages of in VIVO cloning.

A
  • Useful when introducing a gene into another organism. e.e once the gene is in plasmid this plasmid can transfer the gene to humans.
  • Almost no risk of contamination.
  • Very accurate (more than PCR) - mutations are rare.
  • Cuts out specific genes - specific gene (not whole DNA sample).
  • Produces transformed bacteria that can produce large amounts of the protein.
58
Q

Why is there little risk of contamination in in vivo cloning?

A

• Restriction endonuclease match sticky ends to opened up plasmid.

59
Q

What is a DNA probe?

A

A short single stranded length of DNA that has some sort of label attached.

60
Q

What re the two most common types of DNA probe?

A
  • Radioactively labelled - made with nucleotides with a P32 phosphorus isotope.
  • Fluorescently labelled probes - fluoresce under certain conditions.
61
Q

What are DNA probes used for?

A

To identify particular alleles of genes.

62
Q

How does a DNA probe identify a particular allele? (4)

A
  • Complementary base sequence to that of the allele.
  • Double stranded DNA being tested us separated.
  • Separated strands are mixed with probe which binds to complementary bases - DNA hybridisation.
  • The site at which the probe binds can be identified by the radioactivity or fluorescence of the probe.
63
Q

What must be known in order to make a specific DNA probe.

A

The base sequence of the allele.

64
Q

What is DNA hybridisation?

A

When a section of DNA or RNA binds to a single stranded section of DNA with complementary bases.

65
Q

How does DNA hybridisation work? (3)

A
  • Double stranded DNA heated up.
  • Strands Separate
  • Cooled - so strands rejoin however if other complementary DNA is present it is just as likely to form base pairs.
66
Q

How is a specific (mutant) allele for a gene located using DNA hybridisation and DNA probes? (10)

A
  • Determine the base sequence of the allele (genetic library or sequencing)
  • DNA fragment produced complementary to allele.
  • PCR - amplified.
  • Marker attached to make DNA probe.
  • DNA from the person being tested is heated (to separate the two strands).
  • Cooled in a mixture with DNA probes.
  • If the allele is present the DNA probes will binds.
  • DNA is washed.
  • Remaining DNA will now be fluorescently labelled.
  • Dye is detected by shining light onto fragments.
67
Q

Why is it important to screen individuals who may carry a mutant allele?

A

If they are heterozygous, they could have homozygous recessive children. Genetic councillors can advise on the potential implications of having children.

68
Q

How is it possible to test simultaneously for many genetic diseases?

A

Arrange an array of DNA probes on a glass slide and add a DNA sample.

69
Q

How can genetic screening be useful in terms of caner?

A

Detecting for mutations in tumor suppressor genes. If someone inherits one mutated allele they are more at risk of cancer.

70
Q

What is the benefit of screening mutated tumor suppressor genes?

A

• Can get early treatment, check for sings of cancer, change lifestyle etc…

71
Q

How can genetic screening lead to personalised medicine?

A

For different individuals, some drugs be more effective based on their genotype. Or doctors can work out the dose required to be effective for different individuals.

72
Q

What is genetic counselling? What does it allow?

A

Advice and information is given that enable people to make personal decisions e.g. family planning based on genetic disease.

73
Q

After genetic counselling what decisions could be made?

A
  • Whether to have IVF

* Further tests etc..

74
Q

How can eugenic screening help treat cancer more effectively? (3)

A
  • Oncogene mutations -determine the type of cancer - most effective treatment.
  • Gene changes that predict which patients are more likely to benefit from a treatment.
  • Can detect a single cancer cell among millions. Early detection.
75
Q

What are variable number tandem repeats?

A

DNA bases that are non coding. Repeated base sequences.

76
Q

How much of our DNA doesn’t code for a known gene?

A

95%

77
Q

Why are VNTRs used in genetic fingerprinting?

A

Each individual has a unique number and length of VNTRs.

78
Q

What does gel electrophoresis do?

A

Separates DNA fragments according to size.

79
Q

How does gel electrophoresis work?

A
  • DNA fragments placed on agar gel and voltage applied.
  • DNA moves to +ve end.
  • Resistance of the gel means large DNA fragments move furthest.
  • Fragments of different lengths are separated.
  • Labelled with probes - e.g. x-ray film placed over final result.
80
Q

Why does DNA move to the +ve electrode?

A

• DNA is -ve overall (phosphate)

81
Q

Outline the 5 steps of making a genetic fingerprint.

A
  1. Extraction of DNA from sample (PCR)
  2. Digestion using restriction endonuclease.
  3. Separation using gel electrophoresis.
  4. Hybridisation - DNA probes added.
  5. Development
82
Q

Where do the restriction endonucleases cut in genetic fingerprinting?

A

• In the DNA either side of the target VNTR

83
Q

What occurs after separation of strands and before the addition of DNA probes in genetic fingerprinting?

A

Washed with alkali to separate strands.

84
Q

What is the base sequence of DNA probes complementary to in genetic fingerprinting?

A

The VNTR base sequence.

85
Q

How does a computer identify the lengths of DNA on a DNA fingerprint sheet.

A

Compares them to known lengths of DNA run during gel electrophoresis.

86
Q

How are two DNA fingerprints compared?

A

Visually/computer

87
Q

Give 4 uses of DNA fingerprinting.

A
  • Genetic relationships and variability.
  • Forensic Science.
  • Medical diagnosis
  • Plant and animal breeding.
88
Q

Explain how DNA fingerprinting is used to identify whether someone is the genetic father of a child. Give some detail. (2)

A
  • Compare Childs genetic fingerprint to its mother and potential father.
  • Each band from the child should have a corresponding band on one of the two parents as 50% of their DNA is from each parent.
89
Q

Explain how DNA fingerprints can give an idea of the genetic diversity of a population.

A

The more similar the genetic fingerprint the more closely related two organisms are.
If the population has similar fingerprints there is little genetic diversity.

90
Q

Briefly, how can DNA fingerprinting be used in forensic science.

A

Take DNA samples from the crime scene. Compare to suspects.

91
Q

Why might a DNA sample from a crime scene not mean they committed the crime? (4)

A
  • They may have been present but not committed the crime
  • May be from a previous occasion.
  • DNA may belong to a close relative.
  • DNA sample may have been contaminated after the crime. (By the persons DNA or chemicals affecting enzymes)
92
Q

What must be done before DNA can be used to convict someone?

A

Probability must be calculated that someone else DNA matches the suspects.

93
Q

How can genetic fingerprinting be used in medical diagnosis? E.g. Huntington’s where the more repeats of (AGC) at the end of a gene leads to earlier onset of the disease. <30 is unlikely to develop symptoms. >50 early onset likely.

A
  • Fingerprints compared with people who have the disease and those who don’t or those with early onset.
  • Probability of developing symptoms can be determined.
94
Q

How is genetic fingerprinting used in animal breeding?

A
  • Prevent inbreeding
  • Identify desirable alleles.
  • Establishing a pedigree by identifying of paternity.

Biology (18 decks)

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